Stimuli-Responsive Polymer-Prodrug Hybrid Nanoplatform for Multistage siRNA Delivery and Combination Cancer Therapy

Abstract

Nanoparticles (NPs) formulated with cationic lipids and/or polymers have shown substantial potential for systemic delivery of RNA therapeutics such as small interfering RNA (siRNA) for the treatment of cancer and other diseases. While both cationic lipids and polymers have demonstrated the promise to facilitate siRNA encapsulation and endosomal escape, they could also hamper cytosolic siRNA release due to charge interaction and induce potential toxicities. Herein, a unique polymer-prodrug hybrid NP platform was developed for multistage siRNA delivery and combination cancer therapy. This NP system is composed of (i) a hydrophilic polyethylene glycol (PEG) shell, (ii) a hydrophobic NP core made with a tumor microenvironment (TME) pH-responsive polymer, and (iii) charge-mediated complexes of siRNA and amphiphilic cationic mitoxantrone (MTO)-based prodrug that are encapsulated in the NP core. After intravenous administration, the long-circulating NPs accumulate in tumor tissues and then rapidly release the siRNA-prodrug complexes via TME pH-mediated NP disassociation for subsequent tissue penetration and cytosolic transport. With the over-expressed esterase in tumor cells to hydrolyze the amphiphilic structure of the prodrug and thereby induce destabilization of the siRNA-prodrug complexes, the therapeutic siRNA and anticancer drug MTO can be efficiently released in the cytoplasm, ultimately leading to the combinational inhibition of tumor growth via concurrent RNAi-mediated gene silencing and MTO-mediated chemotherapy.

Full text

Stimuli-Responsive Polymer−Prodrug Hybrid Nanoplatform for
Multistage siRNA Delivery and Combination Cancer Therapy
Phei Er Saw,
†,‡
Herui Yao,
†,‡
Chunhao Lin,
†,‡
Wei Tao,
§
Omid C Farokhzad,*
,§
and Xiaoding Xu*
,†,‡
†
Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Medical Research Center, Sun
Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, P. R. China
‡
RNA Biomedical Institute, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou 510120, P. R. China
§
Center for Nanomedicine and Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston,
Massachusetts 02115, United States
*
SSupporting Information
ABSTRACT: Nanoparticles (NPs) formulated with cationic lipids and/or
polymers have shown substantial potential for systemic delivery of RNA
therapeutics such as small interfering RNA (siRNA) for the treatment of cancer
and other diseases. While both cationic lipids and polymers have demonstrated the
promise to facilitate siRNA encapsulation and endosomal escape, they could also
hamper cytosolic siRNA release due to charge interaction and induce potential
toxicities. Herein, a unique polymer−prodrug hybrid NP platform was developed
for multistage siRNA delivery and combination cancer therapy. This NP system is
composed of (i) a hydrophilic polyethylene glycol (PEG) shell, (ii) a hydrophobic
NP core made with a tumor microenvironment (TME) pH-responsive polymer,
and (iii) charge-mediated complexes of siRNA and amphiphilic cationic mitoxantrone (MTO)-based prodrug that are
encapsulated in the NP core. After intravenous administration, the long-circulating NPs accumulate in tumor tissues and then
rapidly release the siRNA−prodrug complexes via TME pH-mediated NP disassociation for subsequent tissue penetration and
cytosolic transport. With the overexpressed esterase in tumor cells to hydrolyze the amphiphilic structure of the prodrug and
thereby induce destabilization of the siRNA−prodrug complexes, the therapeutic siRNA and anticancer drug MTO can be
efficiently released in the cytoplasm, ultimately leading to the combinational inhibition of tumor growth via concurrent RNAi-
mediated gene silencing and MTO-mediated chemotherapy.
KEYWORDS: Nanoparticles, stimuli-responsive, prodrug, multistage siRNA delivery, combination cancer therapy
RNA interference (RNAi) technology has demonstrated
tremendous potential for disease treatment by specific
silencing the expression of target gene(s), especially those
encoding “undruggable”proteins.
1−3
However, RNAi ther-
apeutics such as small interfering RNA (siRNA) are susceptible
to nucleases and cannot readily cross cell membrane due to
their polyanionic and biomacromolecular characteristics.
4−6
Therefore, specific delivery carriers are required to protect
siRNA from enzymatic degradation and to facilitate its cellular
uptake and cytosolic transport. The most commonly used
carriers are cationic lipids and polymers,
7−12
which can bind
siRNA to form complexes (lipoplexes or polyplexes) via
electrostatic interaction. While both cationic lipids and
polymers can improve the siRNA stability, increase its cellular
uptake, and enhance endosomal escape,
13−16
they could also
hamper the cytosolic siRNA release due to charge
interaction.
17−19
More importantly, due to the lower charge
density and molecular weight of siRNA compared to plasmid
DNA, high doses of cationic lipids or polymers are usually used
to increase the siRNA loading ability and stability,
20,21
which in
turn causes severe cytotoxicity to normal tissues.
In the past decade, much effort has been paid to develop safe
and effective siRNA delivery systems. To date, various
innovative approaches, such as the use of coordination
chemistry
21−24
and a guanidine group
15,25−28
with stronger
RNA binding affinity, have been employed to construct siRNA
delivery carries. Nevertheless, stronger RNA binding affinity in
these strategies may also hamper the cytosolic siRNA release.
Although the use of bioresponsive linkages (e.g., disulfide
bond) can endow the carriers with stimuli-responsive
biodegradability,
12,29−32
the generated small cationic molecules
after degradation may still induce potential toxicity and side
effects.
To address these issues, we herein developed a tumor
microenvironment (TME)-responsive polymer−prodrug hy-
brid nanoparticle (NP) platform for multistage siRNA delivery
and combination cancer therapy. Prodrug is a promising
strategy to improve the selectivity and efficacy of a chemo-
Received: April 22, 2019
Revised: August 5, 2019
Published: August 5, 2019
Letter
pubs.acs.org/NanoLett
Cite This: Nano Lett. 2019, 19, 5967−5974
© 2019 American Chemical Society 5967 DOI: 10.1021/acs.nanolett.9b01660
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therapeutic drug.
33,34
By modifying chemotherapeutic drugs
with different ligands, the prodrugs show high stability, long
blood circulation, low immunogenicity, and specific targeting
to tissues or cells.
35−37
More importantly, several anticancer
drugs such as mitoxantrone (MTO) and metformin possess
intrinsic cationic characteristic.
28,38,39
Therefore, rational
modification of these drugs may provide valuable opportunity
to form electrostatic interaction with the negatively charged
siRNA, which not only avoids the potential toxicity of
traditional cationic lipids and polymers but also provides the
feasibility to achieve combination cancer therapy. To this end,
we chose MTO and synthesized an amphiphilic cationic MTO-
based prodrug (Scheme 1A), which can coassemble with a
TME pH-responsive polymer methoxyl-poly(ethylene glycol)-
b-poly(2-(pentamethyleneimino)ethyl methacrylate) (Meo-
PEG-b-PPMEMA)
40,41
to form polymer−prodrug hybrid
NPs. After siRNA encapsulation, the resulting NP platform
shows the following features (Scheme 1B): (i) hydrophilic
PEG chains prolong blood circulation and thereby enhance
tumor accumulation via the enhanced permeability and
retention (EPR) effect; (ii) TME pH-triggered protonation
of the hydrophobic PPMEMA segment induces the rapid NP
disassociation and exposure of siRNA−prodrug complexes that
can penetrate tumor tissues and enhance cytosolic siRNA
transport; (iii) overexpressed esterase in the tumor cells can
hydrolyze the amphiphilic structure of prodrug and thus
induce destabilization of the siRNA−prodrug complexes,
42−44
leading to efficient cytosolic release of siRNA and intact MTO
to accomplish combination cancer therapy.
In this work, Polo-like kinase 1 (PLK1) was chosen as a
therapeutic target and we systematically evaluated the TME
pH-responsive polymer−prodrug hybrid NPs for PLK1 siRNA
(siPLK1) delivery and their anticancer efficacy. PLK1 is a
proto-oncogene that overexpressed in several different cancers
(e.g., breast cancer and prostate cancer)
45−47
and its high
expression is correlated with the low survival rate of
patients.
48−51
Previous studies have demonstrated that PLK1
not only plays an important role in regulating cell mitosis but
also can reduce the therapeutic efficacy of chemotherapeutic
drugs via microtubule rearrangement and DNA damage
recovery.
52−54
Therefore, concurrent down-regulation of
PLK1 expression is expected to improve the therapeutic
efficacy of cancer chemotherapy. Our in vivo results
demonstrate that the TME pH-responsive hybrid NP platform
can efficiently deliver siPLK1 to tumor cells, leading to
combinational inhibition of tumor growth of breast cancer via
concurrent PLK1 silencing and MTO-based chemotherapy.
Results and Discussion. The structure of the MTO-based
prodrug is shown Scheme 1A. MTO is a clinically approved
anticancer drug and has been widely used for the treatment of
acute myelogenous leukemia, breast cancer, and advanced
prostate cancer.
55
Compared to other anticancer drugs (e.g.,
cisplatin and docetaxel), it is convenient to track the MTO
distribution in real-time both in vitro and in vivo, due to its NIR
characteristic with excitation at 610 and 660 nm and emission
at 685 nm.
56−58
More importantly, the cationic nature of MTO
may provide the possibility to complex negatively charged
siRNA for combination cancer therapy. Therefore, to mimic
the typical structure of cationic lipids,
7
two hydrophobic tails
were conjugated to the MTO structure via ester bond to obtain
an amphiphilic cationic MTO-based prodrug (denoted SA-
MTO). Successful synthesis of the prodrug SA-MTO was
confirmed by nuclear magnetic resonance (NMR, Figure S1).
Fluorescence spectroscopy analysis shows that the incorpo-
ration of hydrophobic tails does not affect the NIR
characteristic (Figure 1A), ensuring the feasibility of real-
time tracking the MTO distribution. We first employed high-
performance liquid chromatograph (HPLC) to examine
whether the prodrug can be hydrolyzed by esterase to release
the pharmacologically active MTO. As shown in Figure 1B, the
typical signal corresponding to the intact MTO molecules can
be observed when incubating the prodrug with esterase for 4 h.
By prolonging the incubation time, more intact MTO
molecules can be detected and almost all the prodrug
molecules have been hydrolyzed 12 h later, which could
ensure the pharmacological activity of MTO since esterase has
been demonstrated to be overexpressed in cancer cells.
42−44
After the successful synthesis of the prodrug and validation
of its esterase-triggered hydrolysis, we next synthesized the
TME pH-responsive polymer Meo-PEG-b-PPMEMA (pKa∼
6.89) (Figures S2 and S3),
40,41
which was subsequently
formulated with the prodrug SA-MTO to prepare the
polymer−prodrug hybrid NPs. As shown in Figure 1C, when
mixing the dimethylformamide (DMF) mixture of the prodrug
SA-MTO and polymer Meo-PEG-b-PPMEMA with the siRNA
aqueous solution followed by adding to deionized (DI) water,
self-assembled spherical siRNA-loaded NPs can be formed. In
this polymer−prodrug hybrid NP system, the amphiphilic
cationic prodrug SA-MTO first forms complexes the negatively
Scheme 1. (A) Chemical Structure of the Amphiphilic
Cationic Prodrug SA-MTO and TME pH-Responsive
Polymer Meo-PEG-b-PPMEMA; (B) Schematic Illustration
of the TME pH-Responsive Polymer−Prodrug Hybrid
Nanoplatform for Multistage siRNA Delivery and
Combination Cancer Therapy
a
a
After intravenous injection, the hybrid NPs can extravasate from
leaky tumor vasculature and accumulate in the tumor tissue (a).
Subsequently, the TME pH-triggered NP disassociation induces rapid
release of the siRNA−prodrug complexes (b), which can penetrate
tumor tissue and enter the tumor cells (c). After cellular uptake (d),
the overexpressed esterase in tumor cells can hydrolyze the ester bond
of the prodrug SA-MTO (e), leading to efficient release of therapeutic
siRNA and intact MTO in the cytoplasm, which thereby inhibit the
tumor growth via concurrent RNAi therapy (f) and chemotherapy
(g).
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DOI: 10.1021/acs.nanolett.9b01660
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charged siRNA in the DMF solution via electrostatic
interaction with the hydrophobic tails of the prodrug
positioned on the surface of the complexes. When adding
these complexes to DI water, they can be embedded in the
hydrophobic cores of the self-assembled polymer Meo-PEG-b-
PPMEMA via the hydrophobic interaction with PPMEMA
segment.
31,59−61
We varied the N/P molar ratio by changing
the prodrug amount to adjust the physiochemical properties of
the siRNA-loaded NPs. As shown in Figure 1D, as the N/P
ratio increases from 5 to 30, the resulting NPs (denoted NP5,
NP10, NP15, NP20, and NP30) show increased siRNA
encapsulation efficiency (EE) from ∼35.1% to 68.9% and
particle size from ∼70 to 142 nm. One possible reason is that
the increased N/P ratio induces much stronger electrostatic
interaction with the siRNA, leading to encapsulating more
siRNA−prodrug complexes in the NPs with larger particle size.
Similarly, the ζpotential and drug loading level (LL) of the
resulting NPs increases from NP5 to NP30 as the increased
amount of the prodrug SA-MTO was used.
Since the TME pH-responsive polymer Meo-PEG-b-
PPMEMA was incorporated into the NPs, we next examined
the TME pH response of these NPs. Taking NP15 for
example, when this nanoplatform was incubated in the solution
at a pH below the pKaof the polymer (e.g., pH 6.8), the rapid
protonation of the polymer Meo-PEG-b-PPMEMA induced a
rapid NP dissociation and led to a dramatic decrease in NP
number (Figure 1E). Furthermore, unlike the well-defined NPs
with spherical morphology at pH 7.4, small size particles and
large amorphous aggregates can be observed in the pH 6.8
solution, which is further proven by dynamic light scattering
(DLS) analysis (Figure S4) and may correspond to the
exposed siRNA−prodrug complexes and ionized polymer,
respectively. To further confirm the presence of siRNA−
prodrug complexes after NP dissociation, siRNA labeled with
fluorescein (FL) and its quencher (Dabcyl) were encapsulated
in the NPs and the FL fluorescence at different pHs was
examined.
15,62,63
The naked siRNA can be degraded by RNase
and thus FL and its quencher are separated, leading to a
significant increase in the FL fluorescence (Figure S5). In
contrast, because the cationic prodrug SA-MTO is able to
condense siRNA to form stable complexes that could protect
siRNA from degradation, no obvious change can be seen in the
FL fluorescence although NP dissociation appears at pH 6.8.
Furthermore, this TME pH-triggered rapid NP dissociation
results in a fast siRNA release. As shown in Figure 1F, more
than 60% of the loaded siRNA has been released within 8 h at
pH 6.8, while only around 10% of the loaded siRNA is released
at pH 7.4. Moreover, because the addition of esterase can
hydrolyze the ester bond in the cationic prodrug SA-MTO and
subsequently induce destabilization of the siRNA−prodrug
complexes, the NPs show faster siRNA release than that of the
NPs incubated in the pH 6.8 solution without esterase.
Notably, because the NPs can maintain their structure in the
pH 7.4 solution and prevent the esterase from hydrolyzing the
prodrug SA-MTO encapsulated in the hydrophobic cores,
there is no significant difference in the siRNA release
compared to the NPs incubated in the pH 7.4 solution
without esterase. Similar results can be found in the MTO
release profile (Figure 1G). In the presence of esterase, the
NPs incubated in the pH 6.8 solution show faster MTO release
than that incubated in the pH 7.4 solution. In the absence of
esterase, there is nearly no difference in the free MTO release
Figure 1. (A) Fluorescence emission spectra and fluorescence images of the prodrug SA-MTO and free MTO in the mixture of DMF and
deionized water (8/2, v/v). (B) HPLC profiles of the prodrug SA-MTO and free MTO incubated with esterase for different times. (C) Size
distribution and morphology of the NP15 in aqueous solution at pH 7.4. (D) Size, zeta potential (ζ), siRNA encapsulation efficiency (EE%), and
prodrug loading level (LL%) of the siRNA-loaded NPs prepared at different N/P molar ratios. (E) NP number (count rate) of the NP15 in PBS
buffer at pH 6.8 for different times and the morphology of the NP15 after incubating in PBS buffer at pH 6.8 for 10 min. (F, G) Cumulative siRNA
(F) and free MTO (G) release from the NP15 incubated in PBS buffer at different pHs with or without esterase.
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between these two pHs even the appearance of NP
dissociation at pH 6.8.
We next investigated whether the TME pH-triggered NP
dissociation can enhance the siRNA uptake and improve gene
silencing efficacy. Luciferase-expressing human breast cancer
cells (Luc-MDA-MB-231) were used to incubate with Cy3-
labeled siRNA-loaded NP15 at different pHs for 2 h, and the
siRNA uptake was viewed by a confocal laser-scanning
microscope (CLSM). As shown in Figure 2, the cells show
stronger siRNA uptake at pH 6.8 (Figure 2Ba) than pH 7.4
(Figure 2Aa). Noteworthy, these internalized siRNA molecules
at pH 6.8 are mainly distributed in the cytoplasm, as
demonstrated by colocalization analysis of endosomes and
siRNA molecules (Figure 2Bf). In addition, with the abundant
intracellular esterase to hydrolyze the ester bond of the cationic
prodrug SA-MTO and subsequently induce destabilization of
the siRNA−prodrug complexes, the encapsulated MTO
molecules can be released (Figure 2Be) and enter the nuclei
(Figure 2Bh). The similar result can be found in the cells
treated with the siRNA−prodrug complexes (Figure S6),
indicating that the improved cellular uptake and endosomal
escape is mainly due to the exposed siRNA−prodrug
complexes after TME pH-triggered NP dissociation. The
improved siRNA uptake at pH 6.8 is further quantified by flow
cytometry analysis (Figure 2C,D), in which the siRNA uptake
at pH 6.8 is around 5-fold higher than that of the cells treated
with the siRNA-loaded NPs at pH 7.4.
Having confirmed the improved siRNA uptake induced by
the TME pH-triggered NP dissociation, we then examined the
gene silence efficacy by encapsulating luciferase siRNA (siLuc)
into the NPs. As shown in Figure 2E, all the siLuc-loaded NPs
can down-regulate Luc expression at a 20 nM siRNA dose,
though the silencing efficacy differs depending on the NP
formulation. Moreover, with the improved siRNA uptake at
pH 6.8 (Figure 2Ba,C), the siLuc-loaded NPs offer much
better gene silencing efficacy than that at pH 7.4. Especially for
the NP15 platform, its gene silencing efficacy is close to the
commercial Lipofectamine 2000 (Lipo2K) at pH 6.8. By
replacing the polymer Meo-PEG-b-PPMEMA with non-pH-
responsive polymer methoxyl-poly(ethylene glycol)-b-poly-
(lactic-co-glycolic acid) (Meo-PEG-b-PLGA), there is nearly
no difference in the gene silencing efficacy between pH 7.4 and
6.8, highlighting the importance of TME pH-triggered NP
dissociation to improve the gene silencing. In comparison, the
siLuc-loaded NP15, NP20, and NP30 with higher siRNA
encapsulation efficiency show better silencing efficacy than that
of other NP platforms (NP5 and NP10) with relatively lower
siRNA encapsulation efficiency. Herein, we chose NP15 for the
following experiments since this NP platform shows moderate
ζpotential and smaller particle size (<100 nm).
Figure 2. (A, B) CLSM images of Luc-MDA-MB-231 cells incubated
with the Cy3-labeled siLuc-loaded NP15 at pH 7.4 (A) or 6.8 (B) for
2 h. The endosomes and nuclei were stained with lysotracker green
and Hoechst 33342, respectively. (C, D) Flow cytometry profile (C)
and mean fluorescence intensity (MFI, D) of Luc-MDA-MB-231 cells
incubated with the Cy3-labeled siLuc-loaded NP15 at different pHs
for 2 h. (E) Luc expression in Luc-MDA-MB-231 cells treated with
the Lipo2K/siLuc complexes or the siLuc-loaded NPs at a 20 nM
siRNA dose. For each NP formulation, the NPs loaded scrambled
siRNA were used as control.
Figure 3. (A, B) Western blot analysis of PLK1 expression in MDA-
MB-231 cells treated with the siPLK1-loaded NP15 at pH 7.4 (A) or
6.8 (B). (C) Immunofluorescence analysis of PLK1 expression (red
fluorescence) in MDA-MB-231 cells treated with the siPLK1-loaded
NP15 at a 20 nM siRNA dose. White arrows indicate the
unconsolidated nuclei. (D, E) Flow cytometry analysis (D) and
quantification of apoptosis (E) of MDA-MB-231 cells treated with
siLuc- or siPLK1-loaded NP15 at a 20 nM siRNA dose. (F)
Proliferation profile of MDA-MB-231 cells treated with siLuc- or
siPLK1-loaded NP15 at a 20 nM siRNA dose. The cells incubated
with the blank NPs were used as the control in these experiments.
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DOI: 10.1021/acs.nanolett.9b01660
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After NP screening to obtain the optimal polymer−prodrug
hybrid NP platform (NP15), we next examined the feasibility
of using this RNAi nanoplatform to silence PLK1, a proto-
oncogene that overexpressed in several different cancers
including breast cancer.
46,47,49
As shown in Figure 3A, the
siPLK1-loaded NP15 can down-regulate PLK1 expression in
the MDA-MB-231 cells at pH 7.4 and around 70% knockdown
can be achieved at a 20 nM siRNA dose (Figure S7). More
importantly, due to the improved siRNA uptake at pH 6.8, the
siPLK1-loaded NPs exhibit stronger PLK1 silencing and
around 90% of the PLK1 expression is suppressed at a 20
nM siRNA dose (Figure 3B, Figure S7). Immunofluorescence
staining analysis (Figure 3C) also indicates similar findings.
Compared to the residual PLK1 (red fluorescence) in the cells
treated with the siPLK1-loaded NP15 at pH 7.4, red
fluorescence is nearly absent in the cells treated with the
same NPs at pH 6.8. Due to this efficient PLK1 silencing, the
nuclei become shrunk and even unconsolidated (white arrow
in Figure 3), implying the presence of cell apoptosis or
necrosis. The flow cytometry analysis further proves our
prediction (Figure 3D,E). For the cells treated with the
siPLK1-loaded NP15 at pH 6.8, the percentage of apoptotic
cells reaches around 60%, which is 3- or 2-fold higher than that
of the cells treated with the siLuc- (∼22.5%) or siPLK1-loaded
NP15 (∼38.6%) at pH 7.4, respectively. In addition, the PLK1
silencing also significantly inhibits the proliferation of MDA-
MB-231 cells. As shown in Figure 3F, after 24 h incubation
with the siPLK1-loaded NP15 at pH 6.8 followed by
incubation with fresh culture medium for 6 days, only half of
the cells are alive. However, within the same time frame, there
is about 8- or 4-fold increase in the number of cells treated
with the siLuc- or siPLK1-loaded NP15 at pH 7.4, respectively.
Notably, compared to the cells treated the blank NPs with no
payload (Control), the observed apoptosis (Figure 3D,E) and
inhibited proliferation of the cells treated with the siLuc-loaded
NP15 is mainly due to the loaded SA-MTO prodrug in the
NPs,
57
but not the toxicity of the polymer Meo-PEG-b-
PPMEMA (Figure S8, around 90% cell viability at a polymer
concentration of 100 mg/L). According to the drug loading
level and NP formulation shown in Figure 1D, the final
concentration of MTO and the polymer Meo-PEG-b-
PPMEMA is ∼1.8 and 80 mg/L, respectively, when 20 nM
siRNA dose is used in the in vitro experiments (Figure 3C−F).
Having validated the efficient gene silencing of the hybrid
NP15 platform in vitro,wenextexamineditsin vivo
pharmacokinetics (PK) and biodistribution (BioD). In general,
fluorescent dyes are used to label NPs to facilitate real-time
monitoring of their circulation and distribution in vivo.
However, the use of fluorescent dyes may also affect the
physiochemical properties of the resulting NPs and thus the
obtained results may not reflect the real in vivo behaviors of the
original NPs. Herein, due to the NIR characteristic of MTO,
no fluorescent dye was used to label the hybrid NP15 platform,
which can efficiently avoid the complicated process for dye-
labeling and its potential influence on the physiochemical
properties of the resulting NPs. Figure 4A shows the blood
circulation profile of the siLuc-loaded NP15 after intravenous
(iv) injection to healthy mice (1 nmol of siRNA dose per
mouse, 4.5 mg/kg MTO-equivalent dose, n= 3). Due to the
protection by PEG outer layer,
64−66
the siRNA-loaded NPs
Figure 4. (A) Blood circulation profile of naked siRNA, free MTO, and the siLuc-loaded NP15. (B) Overlaid fluorescence image of the MDA-MB-
231 xenograft tumor-bearing nude mice at 24 h post injection of naked siRNA, free MTO, and the siLuc-loaded NP15. Tumors are indicated by
ellipses. (C) Biodistribution of naked siRNA, free MTO and the siLuc-loaded NP15 in the tumors and major organs of the MDA-MB-231
xenograft tumor-bearing nude mice sacrificed at 24 h postinjection. (D) CLSM images of the 3D tumor spheroids incubated with the siLuc-loaded
NP15 and PLGA NPs (Control NPs) at pH 7.4 or 6.8 for 4 h. (E) Fluorescence images of the MDA-MB-231 tumor sections at 4 h postinjection of
naked siRNA, free MTO, and siLuc-loadedNP15 and PLGA NPs (Control NPs).
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DOI: 10.1021/acs.nanolett.9b01660
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show long blood circulation with a half-life of around 1.02 h. In
contrast, naked siRNA (1 nmol of siRNA dose per mouse) or
free MTO (4.5 mg/kg) is cleared rapidly from the blood and
their blood circulation half-lives are less than 10 min. The
BioD was examined by iv injection of siLuc-loaded NP15 to
the MDA-MB-231 xenograft tumor-bearing mice (1 nmol of
siRNA dose per mouse, 4.5 mg/kg MTO-equivalent dose, n=
3). Figure 4B shows the whole body fluorescence image of the
mice at 24 h post injection. The siRNA-loaded NPs show a
much higher tumor accumulation than naked siRNA or free
MTO. The tumors and major organs (heart, liver, spleen, lung,
and kidney) were harvested (Figure S9) and the BioD was
determined by examining the fluorescence intensity of each
tissue. As shown in Figure 4C, the siRNA-loaded NPs show
more than 7-fold higher tumor accumulation than that of
naked siRNA or free MTO.
With the above promising BioD result, we subsequently
investigated whether the tumor-accumulated NPs can respond
to TME pH to enhance tumor penetration. The 3D tumor
spheroids were first constructed to evaluate the penetration
ability.
67,68
From the CLSM images shown in Figure 4D, red
fluorescence corresponding to the siRNA-loaded NPs is mainly
located on the periphery of the 3D tumor spheroid treated
with the siLuc-loaded NP15 at pH 7.4 for 4 h. In contrast, for
the spheroid treated with the same NPs at pH 6.8, due to the
TME pH-triggered NP disassociation to expose the small size
siRNA−prodrug complexes with strong tissue penetration
ability, red fluorescence can be clearly observed in the interior
area of the 3D tumor spheroid. This result is similar to that of
the 3D tumor spheroid incubated with the siRNA−prodrug
complexes (Figure S10). By replacing the TME pH-responsive
polymer with the Meo-PEG-b-PLGA polymer (denoted
control NPs), there is no obvious difference in the NP
distribution between pH 7.4 and 6.8. To further demonstrate
the improved tumor penetration induced by the TME pH-
triggered NP disassociation, the MDA-MB-231 xenograft
tumor-bearing mice received the iv injection of the siLuc-
loaded NP15, and tumors were collected at 4 h postinjection
and sectioned for CD31 staining.
14,58,69
As shown in Figure 4E,
the exposed siRNA−prodrug complexes from the siRNA-
loaded NPs can extravasate from tumor vessels and deeply
penetrate the extravascular tumor parenchyma. In contrast, due
to the absence of TME pH response, most of the control NPs
(red fluorescence) can be seen in or around the tumor vessels,
with only a small fraction entering the extravascular tumor
parenchyma.
We finally evaluated the in vivo PLK1 silencing efficacy and
anticancer effect of the polymer−prodrug hybrid NP platform.
To examine the in vivo PLK1 silencing, the siPLK1-loaded
NP15 was intravenously injected to the MDA-MB-231
xenograft tumor-bearing mice (1 nmol of siRNA dose per
mouse, 4.5 mg/kg MTO-equivalent dose, n= 3) for three
consecutive days. As shown in Figure 5A,B, the administration
of the siPLK1-loaded NPs induces ∼70% knockdown in the
PLK1 expression. The immunohistochemistry (IHC) staining
analysis also indicates the efficient PLK1 silencing of the
siPLK1-loaded NP15 (Figures 5C). With this promising in vivo
PLK1 silencing, we next examined anticancer effect by iv
injection of the siPLK1-loaded NP15 to the MDA-MB-231
xenograft tumor-bearing mice once every 2 days (1 nmol of
siRNA dose per mouse, 4.5 mg/kg MTO-equivalent dose, n=
5). As shown in Figure 5D−F, after four consecutive injections,
Figure 5. (A)−(C) Western blot (A, B) and IHC analysis (C) of PLK1 expression in the tumor tissues of the MDA-MB-231 xenograft tumor-
bearing nude mice treated with siLuc- (Control NPs) or siPLK1-loaded NP15. *** P< 0.001. (D, E) Tumor size (D) and weight (E) of the MDA-
MB-231 xenograft tumor-bearing nude mice treated with PBS, naked siPKK1, free MTO, and siLuc-, and siPLK1-loaded NP15. The intravenous
injections are indicated by the arrows. ** P< 0.01; *** P< 0.001. (F) Representative photograph of the MDA-MB-231 xenograft tumor-bearing
nude mice in each group at day 18. Tumors are indicated by ellipses. (G, H) TUNEL (G) and H&E (H) staining of the MDA-MB-231 tumor
tissues after systemic treatment in each group. TUNEL-positive apoptotic cells were stained with red fluorescence.
Nano Letters Letter
DOI: 10.1021/acs.nanolett.9b01660
Nano Lett. 2019, 19, 5967−5974
5972

the siPLK1-loaded NPs can significantly inhibit tumor growth
and there is less than 2-fold increase (from ∼69 to ∼137 mm3)
in tumor size within 18 days. In contrast, for the mice treated
with PBS, naked siPLK1 (1 nmol of siRNA dose per mouse),
or free MTO (4.5 mg/kg), they show more than 7-fold
increase in their tumor size and tumor weight. Due to the
encapsulation of the prodrug SA-MTO in the siLuc-loaded
NPs, they are also able to inhibit tumor growth (>4-fold
increase in the tumor size within 18 day evaluation period).
However, the tumor inhibition efficacy is lower than that of the
siPLK1-loaded NPs showing the characteristic of combination
therapy, i.e., RNAi therapy from siPLK1 and chemotherapy
from MTO. Histological analysis of tumor slides further
demonstrates the fact that the siPLK1-loaded NPs are the most
effective in inducing cell apoptosis (Figure 5G) and inhibiting
cell proliferation (Figure 5H). Moreover, the administration of
siPLK1-loaded NPs does not affect the mouse body weight
(Figure S11). Histological analysis (Figure S12) shows that
there are no obvious histological changes in the main organs
(heart, liver, spleen, lung, and kidney). To further assess the in
vivo safety, the siPLK1-loaded NP15 was administrated to the
healthy mice (1 nmol of siRNA dose per mouse, 4.5 mg/kg
MTO-equivalent dose, n= 3) via iv injection. Immune
response analysis shows that the level of representative
cytokines (TNF-α, IFN-γ, IL-6, and IL-12) is in the normal
range (Figure S13). Blood routine analysis indicates that
aspartate aminotransferase (AST), alanine aminotransferase
(ALT), alkaline phosphatase (ALKP), blood urine nitrogen
(BUN), creatinine, and total protein are in the normal range
(Figure S14). Taken together, these results demonstrate good
biocompatibility of our newly developed polymer−prodrug
hybrid NP platform.
Conclusion. In summary, we have developed a new TME
pH-responsive polymer−prodrug hybrid NP platform for
multistage siRNA delivery and combination cancer therapy.
This long-circulating NP platform can first accumulate in the
tumor tissues and then rapidly respond to TME pH to expose
the small size siRNA−prodrug complexes, which subsequently
penetrate tumor tissues and enter the tumor cells. With the
overexpressed esterase in the tumor cells to break the prodrug
structure, the therapeutic siRNA and anticancer drug can be
efficiently released in the cytoplasm, inducing combinational
inhibition of tumor growth via concurrent RNAi therapy from
siPLK1 and chemotherapy from MTO. This hybrid NP
platform could be used as an effective vehicle for the systemic
delivery of various biomacromolecules (e.g., nucleic acids and
proteins) for cancer therapy.
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.nano-
lett.9b01660.
Materials and methods, synthesis and characterization of
the SA-MTO prodrug, polymers and NPs, body weight
of mice, histology, and immune response; NMR and
fluorescence emission spectra, acid−base titration
profile, size distribution profile, CLSM images, PLK1
expression, cell viability and body weight graphs,
fluorescence image of the tumors and main organs,
histological section of the major organs, serum level
graphs (PDF)
■AUTHOR INFORMATION
Corresponding Authors
*E-mail: ofarokhzad@bwh.harvard.edu.
*E-mail: xuxiaod5@mail.sysu.edu.cn.
ORCID
Wei Tao: 0000-0002-4277-3728
Omid C Farokhzad: 0000-0003-2009-270X
Xiaoding Xu: 0000-0002-9785-6731
Author Contributions
P.E.S., H.Y., and C.L. contributed equally to this work.
Notes
Theauthorsdeclarethefollowingcompetingfinancial
interest(s): O.C.F. has financial interests in Selecta Bio-
sciences, Tarveda Therapeutics, Placon Therapeutics, and Seer.
■ACKNOWLEDGMENTS
This work was supported by the National Natural Science
Foundation of China (81874226 and 81803020), the Interna-
tional Scientific and Technological Cooperation Program from
Guangdong Science and Technology Department
(2018A050506033), the Thousand Talents Program for
Distinguished Young Scholars, the grants from Guangzhou
Science and Technology Bureau (201902020015 and
201704020131) and Guangdong Science and Technology
Department (2017B030314026), the “Three million for Three
Years”Project of the High-level Talent Special Funding
Scheme of Sun Yat-Sen Memorial Hospital, the David H.
Koch-Prostate Cancer Foundation (PCF) Program in Cancer
Nanotherapeutics, and the US METAvivor Early Career
Investigator Award (2018A020560).
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